Xanthomonas Pseudomonas syringae and Ralstonia solanacearum · genes (X. Li, Kapos, and Zhang 2015; Jones, Vance, and Dangl 2016; Kapos, Devendrakumar, and Li 2019). While effector

1

Title

Roq1 confers resistance to Xanthomonas, Pseudomonas syringae and Ralstonia solanacearum

in tomato

Authors

Nicholas C. Thomas1,2, Connor G. Hendrich3, Upinder S. Gill4, Caitilyn Allen3, Samuel F. Hutton4,

*Alex Schultink1,2

Affiliations

1. Fortiphyte Inc., 2151 Berkeley Way, Berkeley, CA 94704

2. Innovative Genomics Institute, University of California, Berkeley, CA 94720

3. Department of Plant Pathology, University of Wisconsin – Madison, Madison, WI 53706, USA

4. University of Florida, IFAS, Gulf Coast Research and Education Center, Wimauma, FL, USA

Author contributions1

Funding information2

Corresponding author contact3

1 N.C.T. and A.S. wrote the manuscript and performed Pseudomonas and Ralstonia petiole infection

assays. A.S. carried out Xanthomonas infection and Agrobacterium transient expression experiments. U.S.G. and S.F.H. performed Xanthomonas field experiments. C.G.H. constructed the Ralstonia knockout and performed Ralstonia soil soak assays, supervised by C.A. All authors analyzed results, edited and approved the manuscript. 2This work was supported in part by the National Institute of Food and Agriculture, US Department of

Agriculture under award number 2016-67012-25106 and the UC Berkeley Innovative Genomics Institute. CGH was supported by an NSF Predoctoral Fellowship. 3 Alex Schultink, [email protected]

.CC-BY-NC 4.0 International licensecertified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which was notthis version posted January 20, 2020. . https://doi.org/10.1101/813758doi: bioRxiv preprint

https://doi.org/10.1101/813758

http://creativecommons.org/licenses/by-nc/4.0/

2

Summary

A single immune receptor expressed in tomato confers strong resistance to three

different bacterial diseases.

Abstract

Xanthomonas species, Pseudomonas syringae and Ralstonia solanacearum are bacterial plant

pathogens that cause significant yield loss in many crop species. Current control methods for

these pathogens are insufficient but there is significant potential for generating new disease-

resistant crop varieties. Plant immune receptors encoded by nucleotide‐binding, leucine‐rich

repeat (NLR) genes typically confer resistance to pathogens that produce a cognate elicitor,

often an effector protein secreted by the pathogen to promote virulence. The diverse sequence

and presence / absence variation of pathogen effector proteins within and between pathogen

species usually limits the utility of a single NLR gene to protecting a plant from a single

pathogen species or particular strains. The NLR protein Recognition of XopQ 1 (Roq1) was

recently identified from the plant Nicotiana benthamiana and mediates perception of the effector

proteins XopQ and HopQ1 from Xanthomonas and P. syringae respectively. Unlike most

recognized effectors, alleles of XopQ/HopQ1 are highly conserved and present in most plant

pathogenic strains of Xanthomonas and P. syringae. A homolog of XopQ/HopQ1, named RipB,

is present in many R. solanacearum strains. We found that Roq1 also mediates perception of

RipB and confers immunity to Xanthomonas, P. syringae, and R. solanacearum when

expressed in tomato. Strong resistance to Xanthomonas perforans was observed in three

seasons of field trials with both natural and artificial inoculation. The Roq1 gene can therefore

be used to provide safe, economical and effective control of these pathogens in tomato and

other crop species and reduce or eliminate the need for traditional chemical controls.


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Introduction

Bacterial pathogens from the species Pseudomonas syringae, Ralstonia solanacearum, and the

genus Xanthomonas can infect many different crop species and inflict significant yield losses

when environmental conditions favor disease. Xanthomonas and P. syringae tend to enter plant

stem, leaf, or flower tissue through wounds or natural openings, such as stomata or

hydathodes, whereas R. solanacearum is soilborne, entering roots through wounds and natural

openings before colonizing xylem tissue (Vasse, Frey, and Trigalet 1995; Gudesblat, Torres,

and Vojnov 2009). Once inside the host these bacteria manipulate host metabolism and

suppress plant immunity using multiple strategies, including effector proteins delivered by the

type III secretion system (Kay and Bonas 2009; Peeters et al. 2013; Xin, Kvitko, and He 2018).

This enables the pathogens to multiply to high titers while the plant tissue is still alive and

showing few or no visual symptoms. Once the bacteria reach high populations they typically

cause necrosis of infected leaf tissue or wilting and eventual death of the plant.

Effective control measures for bacterial pathogens are relatively limited, particularly once plants

become infected (Davis et al. 2013). Soil fumigation can reduce R. solanacearum populations in

the soil but this is expensive, potentially hazardous to workers and the environment, and of

limited efficacy (Yuliar, Nion, and Toyota 2015). Copper sulfate and antibiotics such as

streptomycin have been used to control Xanthomonas species and P. syringae but have

adverse environmental impacts and many strains have evolved tolerance to these chemicals

(Kennelly et al. 2007; Griffin et al. 2017). Applying chemicals that induce systemic acquired

resistance, such as acibenzolar-S-methyl, can provide partial control but increase production

cost and can depress crop yields when used repeatedly (Pontes et al. 2016).

The most effective, economical, and safe way to control bacterial pathogens is to plant crop

varieties that are immune to the target pathogen (Jones et al. 2014; Vincelli 2016). Such


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immunity is often mediated by plant immune receptor genes. Plants have large families of cell

surface and intracellular immune receptor proteins that surveil for the presence of invading

pathogens (Zipfel 2014; Jones, Vance, and Dangl 2016). Effector proteins delivered by the

bacterial type III secretion system are common ligands for intracellular plant immune receptors

encoded by intracellular nucleotide‐binding domain and leucine‐rich repeat containing (NLR)

genes (X. Li, Kapos, and Zhang 2015; Jones, Vance, and Dangl 2016; Kapos, Devendrakumar,

and Li 2019). While effector proteins contribute to virulence on a susceptible host, an immune

response is activated in the plant if that plant has the cognate receptor to recognize the effector.

NLR genes typically confer strong, dominant resistance to pathogens that deliver the cognate

recognized effector protein (Jones and Dangl 2006; Boller and He 2009; Deslandes and Rivas

2012; X. Li, Kapos, and Zhang 2015). Disease-resistant plants can be generated by identifying

the appropriate plant immune receptor genes and transferring them into the target crop species

(Dangl, Horvath, and Staskawicz 2013).

We recently identified the Nicotiana benthamiana immune receptor gene named Recognition of

XopQ 1 (Roq1), which appears to be restricted to the genus Nicotiana and is required for

resistance to Xanthomonas spp. and P. syringae (Schultink et al. 2017). Roq1 is a

Toll/Interleukin‐1 Receptor (TIR) NLR immune receptor that mediates recognition of the

Xanthomonas effector protein XopQ and the homologous effector HopQ1 from P. syringae.

XopQ is present in most species and strains of Xanthomonas (Ryan et al. 2011) and HopQ1 is

present in 62% (290 of 467) sequenced putative pathogenic P. syringae strains (Dillon et al.

2019). XopQ/HopQ1 has homology to nucleoside hydrolases and has been shown to enhance

virulence on susceptible hosts (Ferrante and Scortichini 2009; W. Li et al. 2013), possibly by

altering cytokinin levels or interfering with the activity of host 14-3-3 proteins (W. Li et al. 2013;

Giska et al. 2013; Teper et al. 2014; Hann et al. 2014). The conservation of XopQ/HopQ1 and

their importance in virulence suggests that Roq1 has widespread potential to confer resistance

to these pathogens in diverse crop species. Indeed, transient expression assays demonstrated


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that Roq1 can recognize XopQ/HopQ1 alleles from Xanthomonas and P. syringae pathogens of

tomato, pepper, rice, citrus, cassava, brassica, and bean (Schultink et al. 2017). However, it

was not known if Roq1 can confer disease resistance when expressed in a crop plant.

Tomato is one of the most important vegetable crops and is highly susceptible to several

bacterial diseases. Bacterial spot, bacterial speck and bacterial wilt of tomato are caused by

Xanthomonas species, P. syringae pv. tomato and R. solanacearum, respectively. These

diseases are difficult to control, especially if the pathogens become established in a field and

environmental conditions favor disease (Rivard et al. 2012; Potnis et al. 2015). Tomato breeding

germplasm has only limited resistance against these diseases and in some cases linkage drag

has complicated introgression of resistance genes from wild relatives (Sharma and Bhattarai

2019). R. solanacearum contains a homolog of XopQ/HopQ1 called RipB, suggesting that

expressing Roq1 in tomato could also confer resistance to bacterial wilt. Like XopQ/HopQ1 in

Xanthomonas and P. syringae, RipB is highly conserved in most R. solanacearum isolates

(Sabbagh et al. 2019). Here we present laboratory and field data showing that Roq1 confers

resistance against these three pathogens in tomato and that this resistance depends on the

presence of the cognate pathogen effector.

Results

Tomatoes expressing Roq1 are resistant to Xanthomonas and P. syringae

We generated homozygous tomato plants expressing the Roq1 gene from N. benthamiana and

tested them for resistance to Xanthomonas and P. syringae by measuring bacterial growth in

planta. Population sizes of wild-type X. perforans strain 4B and X. euvesicatoria strain 85-10

were approximately 100-fold smaller in tomatoes expressing Roq1 compared to wild-type

tomatoes at six days post inoculation (Fig. 1). In contrast, XopQ deletion mutants multiplied

equally well in leaves of both wild-type and Roq1 tomato. Disease symptoms begin as small


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water-soaked lesions and progress to necrosis of infected tissue. Wild-type X. perforans and X.

euvesicatoria caused severe disease symptoms on wild-type tomato plants but failed to cause

visible symptoms on Roq1 plants (Fig. 2). The XopQ mutants caused similar disease symptoms

on both wild-type and Roq1 tomato. Similar results were observed for P. syringae DC3000 and

its HopQ1 mutant (Fig. 1, 2) and a Race 1 isolate of P. syringae pv. tomato (Supplementary Fig.

1).

Expression of Roq1 confers resistance to Xanthomonas perforans in the field

To determine if the resistance observed in growth chamber experiments would hold up under

commercial tomato production conditions, we tested the ability of Roq1 tomatoes to resist X.

perforans infection in the field. Roq1 tomatoes were grown along with the Fla. 8000 wild-type

parent as well as a Fla. 8000 variety expressing the Bs2 gene from pepper as a resistant control

(Kunwar et al. 2018). For each of the three growing seasons both Roq1 and the resistant Bs2

control tomatoes showed significantly lower disease severity than the parental Fla. 8000 variety

(Table 1) (p < 0.05). The total marketable yield of the Roq1 plants was not significantly different

from that of the susceptible parent for any of the three seasons (p > 0.05).

The R. solanacearum RipB effector, a homolog of XopQ/HopQ1, is recognized by Roq1

RipB, considered a “core” effector of R. solanacearum, is present in approximately 90% of

sequenced strains (Sabbagh et al. 2019), making it an attractive target ligand for engineering

crop plants to be resistant to this pathogen. Roq1 perceives diverse alleles of XopQ and HopQ1

and we hypothesized that it can also recognize RipB. We constructed a phylogenetic tree of

RipB, XopQ, and HopQ1 alleles identified by BLAST search and observed two major clades of

RipB proteins, corresponding to phylotypes I and III (strains originating in Asia and Africa) and

to phylotype II (strains originating in the Americas) (Fig. 3). We selected RipB alleles from R.

solanacearum strains GMI1000 and MolK2, which are present in clades 1 and 2, respectively,


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for subsequent analysis. These two RipB alleles share 71% amino acid identity with each other

and approximately 52% identity with XopQ excluding the divergent N terminus containing the

putative type III secretion signal. An alignment of these two RipB proteins with XopQ and

HopQ1 is shown in Supplementary Fig. 2. To test for Roq1-dependent recognition of RipB, we

used Agrobacterium to transiently express RipB from GMI1000 and Molk2 in leaf tissue of wild-

type and roq1 mutant N. tabacum. Both RipB alleles triggered a strong hypersensitive / cell

death response in wild-type N. tabacum, indicating immune activation. This response was

absent in the roq1-1 mutant but could be restored by transiently expressing Roq1 along with

XopQ, RipBGMI1000, or RipBMolk2 (Fig. 4).

Roq1 tomatoes are resistant to R. solanacearum containing RipB

Our observation that Roq1 can recognize RipB in leaf transient expression assays suggested

that Roq1 can mediate resistance to bacterial wilt caused by R. solanacearum. We tested this

hypothesis by challenging wild-type and Roq1-expressing tomato plants with R. solanacearum

strain GMI1000 using a soil soak inoculation disease assay. Wild-type plants developed severe

wilting approximately seven days after inoculation, whereas Roq1 tomato plants remained

mostly healthy over the two-week time course. The Roq1 tomato plants were susceptible to a

deletion mutant lacking RipB (GMI1000 ∆ripB) (Fig. 5a). We also challenged plants by

introducing bacteria directly to the xylem by placing bacteria on the surface of a cut petiole.

Wild-type plants were wilted at eight days whereas Roq1 plants remained healthy (Fig. 5b).

Tomatoes expressing Roq1 were also resistant to R. solanacearum strain UW551, which is a

Race 3 Biovar 2 potato brown rot strain that has a clade 2 RipB allele (Supplementary Fig. 3).

Occurrence of RipB in the R. solanacearum species complex


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To investigate the potential for using Roq1 to protect plants from R. solanacearum, we

investigated the occurrence of RipB alleles in select R. solanacearum strains. Table 2

summarizes published known hosts of several R. solanacearum strains along with the phylotype

and identified RipB allele. All strains in Table 2 except for tobacco pathogenic strains K60, Y45,

BK1002 and OE1-1 contain putative full-length and functional RipB alleles. Relative to other

RipB alleles, the K60 RipB allele is truncated at residue 437 and missing approximately 65 C-

terminal residues and the OE1-1 allele is truncated at residue 425, missing approximately 77

residues based on a published genome sequence (Hayes, MacIntyre, and Allen 2017). Y45

does not have a predicted RipB allele based on a draft genome sequence (Z. Li et al. 2011).

Discussion

Roq1 expression in tomato confers strong resistance to X. perforans, X. euvesicatoria and P.

syringae pv. tomato. Its effectiveness is dependent on the presence of the recognized effector

protein XopQ/HopQ1 (Fig. 1, 2). Field trials revealed that tomatoes expressing Roq1 were less

susceptible to X. perforans than wild-type tomatoes in conditions approximating commercial

production (Table 1). Roq1 conferred a similar level of resistance as the Bs2-containing

resistant check variety in one season and was slightly weaker in the other two. Bacterial spot

caused by X. perforans can cause lesions on fruits, making them unsuitable for commercial

sale, and also reduce plant productivity by damaging leaf tissue. Despite showing strong

disease resistance, the tested Roq1 line did not give a significantly greater total marketable

yield than the susceptible parental variety. Of the three seasons, Spring 2019 had weather

conditions expected to be most conducive for observing an impact of bacterial spot on

marketable yield with mid-season rain promoting the early development of disease symptoms.

The average marketable yield for the Roq1 tomatoes was 27% higher than wild-type in this

season, although a relatively small sample size (4 replicate plots of ten plants each) and a large


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variability of yield between plots resulted in a p-value of 0.08 by Student’s t-test. Although we

cannot conclude that Roq1 improves marketable yield of tomatoes from this data, a larger trial

under high disease pressure may reveal such a difference.

It was unclear if Roq1 could confer resistance to R. solanacearum because it colonizes different

tissues than Xanthomonas and P. syringae. While Xanthomonas and P. syringae colonize

tomato leaf tissue, R. solanacearum enters through the roots and colonizes xylem vessels.

Although R. solanacearum’s type III secretion system is essential for virulence, it is not clear

when and where the pathogen delivers effectors into host cells. It was therefore not clear if

Roq1 would be able to confer resistance to this pathogen in tomato. Here we confirmed that

tomato plants expressing Roq1 had strong resistance to R. solanacearum expressing RipB as

measured by both soil soak and cut-petiole inoculation assays. This result is consistent with and

expands on the recent report that RipB is recognized in Nicotiana species and that silencing

Roq1 confers susceptibility to R. solanacearum in Nicotiana benthamiana(Nakano and

Mukaihara 2019). In addition, Roq1 confers resistance to R. solanacearum Race 3 biovar 3

strain UW551, a pathogen that can overcome other known sources of bacterial wilt resistance in

tomato(Milling, Babujee, and Allen 2011). Some but not all of the Roq1 tomatoes inoculated by

the soil soak assay were colonized by R. solanacearum (Supplementary Fig. 4), implying that

Roq1 both restricts the establishment of vascular colonization and separately reduces bacterial

titers if colonization does occur. Activation of immune receptors, including Roq1, is known to

induce many defense-associated genes with different putative activities (Sohn et al. 2014; Qi et

al. 2018), presumably acting to inhibit pathogen virulence by distinct mechanisms. The

observation that Roq1 inhibits both colonization establishment and population growth suggest

that at least two independent downstream defense responses mediate the observed resistance

phenotype.

The Roq1 tomatoes were fully susceptible to an R. solanacearum mutant lacking RipB,

indicating that the resistance depends on the interaction between RipB and Roq1. This is


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consistent with the observation that several naturally occurring R. solanacearum strains that can

infect tobacco have truncated or are missing the RipB effector (Table 2)(Nakano and Mukaihara

2019), suggesting that losing RipB can allow the pathogen to overcome the native Roq1 gene

present in Nicotiana tabacum. Tobacco-infecting strains K60 and OE1-1 contain independently

truncated RipB alleles (Fig. 3) and there have likely been multiple independent gene loss events

which enable strains to evade Roq1-mediated resistance. Similarly, HopQ1 has been lost in

strains of P. syringae that can infect tobacco (Denny 2006; Ferrante and Scortichini 2009; Z. Li

et al. 2011). This suggests that this effector is not essential for virulence and it would therefore

be prudent to deploy Roq1 in combination with other disease resistance traits to avoid

resistance breakdown due to pathogens losing XopQ/HopQ1/RipB.

No other known NLR immune receptor confers resistance against such a broad range of

bacterial pathogens as Roq1. Effectors that are recognized by NLR proteins act as avirulence

factors and are under strong evolutionary pressure to diversify or be lost to evade immune

activation. Therefore the effector repertoires of pathogens are often quite diverse, with relatively

few “core” effectors conserved within a species and even fewer shared between different

genera (Grant et al. 2006). Effectors recognized by plant NLRs are typically narrowly conserved

within a single bacterial genus (Kapos, Devendrakumar, and Li 2019). One such effector is

AvrBs2, recognized by the Bs2 receptor from pepper, which is present in many Xanthomonas

strains but is absent from P. syringae and R. solanacearum. In contrast XopQ/HopQ1/RipB is

highly conserved in most Xanthomonas, P. syringae and R. solanacearum strains that cause

disease in crop plants including kiwi (P. syringae pv. actinidae), banana (R. solanacearum and

X. campestris pv. musacearum), stone fruit (P. syringae), pepper (X. euvesicatoria), citrus (X.

citri), strawberry (X. fragariae), brassica (X. campestris), rice (X. oryzae), potato (R.

solanacearum) and others. R. solanacearum Race 3 Biovar 2 strains are of particular concern

because they are cold tolerant and potentially threaten potato cultivation in temperate climates.


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As a result, R. solanacearum Race 3 biovar 2 is a strictly regulated quarantine pathogen in

Europe and North America and is on the United States Select Agent list. The ability of Roq1 to

protect tomato from the Race 3 Biovar 2 strain UW551 (Supplementary Fig. 3) suggests that

Roq1 can also protect potato from this high-concern pathogen. This work demonstrates the

widespread potential of using naturally occurring plant immune receptors to manage diverse

and difficult to control pathogen species safely, sustainably and economically.

Methods

Generation of tomato expressing Roq1

The Roq1 coding sequence was amplified from N. benthamiana cDNA and cloned into the

pORE E4 binary plasmid (Coutu et al. 2007). A. tumefaciens co-cultivation was used to

transform Roq1 into the commercial tomato variety Fla. 8000 at the University of Nebraska Plant

Transformation Core Research Facility. Transformed plants were selected by resistance to

kanamycin, confirmed by genotyping, and selfed to obtain homozygous lines.

Bacterial Leaf Spot and Leaf Speck disease assays

Xanthomonas spp. cultures were grown in NYG broth (0.5% peptone, 0.3% yeast extract, 2%

glycerol) with rifampicin (100 μg / mL) overnight at 30 °C. P. syringae cultures were grown in KB

broth (1% peptone, 0.15% K2HPO4, 1.5% glycerol, 5 mM MgSO4, pH 7.0) with rifampicin (100

μg / mL) overnight at 28 °C. Bacterial cultures were spun down at 5200 g, washed once with 10

mM MgCl2, and then diluted to the appropriate infiltration density with 10 mM MgCl2. Leaf tissue

of tomato plants (approximately four weeks old) was infiltrated with bacterial solution using a

needleless syringe. To quantify bacterial growth, leaf punches were homogenized in water,

serially diluted and plated on NYG (for Xanthomonas spp.) or KB (for P. syringae) plates

supplemented with 100 μg / mL rifampicin and 50 μg / mL cycloheximide to measure colony


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forming units. X. perforans strain 4B, X. euvesicatoria strain 85-10, and P. syringae strain

DC3000 and the corresponding XopQ/HopQ1 deletion mutants were described previously

(Schwartz et al. 2015; Schultink et al. 2017). The P. syringae pv. tomato Race 1 strain was

isolated from a field of tomatoes with the PTO resistance gene in 1993 in California.

Transient expression of RipB and XopQ

Alleles of RipB from R. solanacearum (NCBI Genbank accessions CAD13773.2 and

WP_003278485) were synthesized and cloned into a BsaI-compatible version of the pORE E4

vector (Coutu et al. 2007). This plasmid was transformed into A. tumefaciens strain C58C1. A.

tumefaciens cultures were grown on a shaker overnight at 30 °C in LB broth with rifampicin (100

μg / mL), tetracycline (10 μg / mL) and kanamycin (50 μg / mL). The cells were collected by

centrifugation and resuspended in infiltration buffer (10 mM 2-(N-morpholino)ethanesulfonic

acid, 10 mM MgCl2, pH 5.6), and diluted to an OD600 of 0.5 for infiltration into N. tabacum leaf

tissue.

N. tabacum roq1 mutant lines

N. tabacum roq1 mutant lines were generated by transforming N. tabacum with a construct

coding for CAS9 and a guide RNA targeting the Roq1 gene with the sequence

GATGATAAGGAGTTAAAGAG. This construct was also used for the generation of N.

benthamiana roq1 mutants published in Qi et al. 2018. Transformed N. tabacum plants were

generated by Agrobacterium co-cultivation and selected for using kanamycin. Transformed

plants were genotyped for the presence of mutations at the target site by PCR and Sanger

sequencing. The N. tabacum mutant roq1-1 has a single base pair A insertion at the target cut

site in the Roq1 gene.

Bacterial wilt virulence assays


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R. solanacearum virulence on tomato was measured as previously described(Khokhani et al.

2018). Briefly, cells of R. solanacearum strain GMI1000 grown overnight in CPG (0.1% casein

hydrolysate, 1% peptone, 0.5% glucose, pH 7.0) at 28°C were collected by centrifugation and

diluted to an OD600 of 0.1 in water (1x108 CFU/ml). 50 mL of this suspension was poured on the

soil around 17-day old tomato plants. Disease was rated daily for two weeks on a 0-4 disease

index scale, where 0 is no leaves wilted, 1 is 1-25% wilted, 2 is 26-50% wilted, 3 is 51-75% of

wilted, and 4 is 76-100% wilted. Data represent a total of four biological replicates with ten

plants per replicate. Virulence data were analyzed using repeated measures ANOVA (Khokhani

et al 2018). For petiole infection, the petiole of the first true leaf was cut with a razor blade

horizontally approximately 1 cm from the stem. A drop of bacterial solution (2 uL, OD600 = 0.001)

was pipetted onto the exposed cut petiole surface.

Field trial disease assays

Three field trials were conducted at the University of Florida Gulf Coast Research and

Education Center in Balm during the spring seasons of 2018 and 2019 and the fall season of

2018 and under the notification process of the United States Department of Agriculture. Large-

fruited, fresh market tomato lines were used in these trials and included the inbred line, Fla.

8000, and nearly-isogenic lines containing either Roq1 (event 316.4) or Bs2 (Kunwar et al.

2018); along with commercial hybrids Florida 91, Sanibel, and HM 1823 as additional,

susceptible controls (data not shown). For each trial, seeds were sown directly into peat-lite

soilless media (Speedling, Sun City, FL, USA) in 128-cell trays (38 cm3 cell size). Transplants

were grown in a greenhouse until 5 or 6 weeks, then planted to field beds that had been

fumigated and covered with reflective plastic mulch. Field trials were conducted using a

randomized complete block design with four blocks and 10-plant plots. Field plants were staked

and tied, and irrigation was applied through drip tape beneath the plastic mulch of each bed. A

recommended fertilizer and pesticide program were followed throughout the growing season,


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excluding the use of plant defense inducers, copper, or other bactericides (Freeman et al.

2018). Fruit were harvested from the inner 8 plants of each plot at the breaker stage and

beyond graded for marketability according to USDA specifications. Yield data were analyzed

using the PROC GLIMMIX procedure in SAS (version 9.4; SAS Institute, Cary, NC, USA), and

block was considered random effects.

Field trials were inoculated with X. perforans race T4 (strain mixture of GEV904, GEV917,

GEV1001, and GEV1063). Bacterial strains were grown on nutrient agar medium (BBL, Becton

Dickinson and Co., Cockeysville, MD) and incubated at 28 °C for 24 h. Bacterial cells were

removed from the plates and suspended in a 10 mM MgSo4·7H2O solution, and the suspension

was adjusted to OD600=0.3, which corresponds to 108 CFU/ml. The suspension for each strain

was then diluted to 106 CFU/ml, mixed in equal volume, and applied along with polyoxyethylene

sorbitan monolaurate (Tween 20; 0.05% [vol/vol]) for field inoculation. Field trial plants were

inoculated approximately 3 weeks after transplanting.

Bacterial spot disease severity was recorded three to eight weeks after inoculation using the

Horsfall-Barratt scale (Horsfall, JG and Barrat, RW 1945), and ratings were converted to

midpoint percentages for statistical analysis. Disease severity data were analyzed using a

nonparametric procedure for the analysis of ordinal data (Brunner and Puri 2001; Shah and

Madden 2004). Analysis of variance type statistic of ranked data was conducted using the

PROC MIXED procedure in SAS. Relative marginal effects (RME) were generated with the

equation: RME = (R – 0.5)/N; where R is the mean treatment ranking, and N is the total number

of experimental units in the analysis; the LD_CI macro was used to generate 95% confidence

intervals (Brunner and Puri 2001; Shah and Madden 2004). Blocks were considered random

effects.

Generation of the R. solanacearum ∆ripB mutant


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An unmarked ∆ripB mutant was created using sacB positive selection with the vector pUFR80

(Castañeda et al. 2005). Briefly, the regions upstream and downstream of ripB were amplified

using the primers ripBupF/R and ripBdwnF/R. These fragments were inserted into pUFR80

digested with HindIII and EcoRI using Gibson Assembly (Gibson et al. 2009) (New England

Biolabs, Ipswitch, MA) and this construct was incorporated into the genome of strain GMI1000

using natural transformation, with successful integrants selected on CPG + kanamycin (Coupat

et al. 2008). Plasmid loss was then selected for on CPG plates containing 5% w/v sucrose.

Correct deletions were confirmed using PCR and sequencing.

Phylogenetic analysis of XopQ, HopQ1 and RipB alleles

RipB alleles were identified by BLAST search of the NCBI protein database. Clustal Omega

(Sievers et al. 2011) was used to generate a multiple sequence alignment with XopQ and

HopQ1 alleles. To span the diversity of RipB alleles without have many redundant sequences,

only a single sequence was retained if there were multiple identical or nearly identical

sequences identified. A maximum likelihood tree was generated using PhyML (Guindon et al.

2010).

Acknowledgements

We thank Shirley Sato and Tom Clemente of the University of Nebraska Plant Transformation

Core Research Facility for the transformation of tomato. We thank Myeong-Je Cho and Julie

Pham of the UC Berkeley Innovative Genomics Institute for transformation of Nicotiana

tabacum.

Competing Interests


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A. S. and N.C.T. are employees of and have a financial stake in Fortiphyte Inc., which has

intellectual property rights related to the Roq1 resistance gene.

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0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

10.0

Figure 1. Bacterial growth in tomatoes

expressing Roq1. Xanthomonas perforans 4B

(Xp), Xanthomonas euvesicatoria 85-10 (Xe), and

Pseudomonas syringae DC3000 (Ps) were

infiltrated into leaf tissue of wild-type tomato and

tomato expressing Roq1 at a low inoculum

(OD600 = 0.0001 for Xe and Xp; OD600 = 0.00005

for Ps). Bacterial abundance was quantified by

homogenizing leaf punches and counting colony

forming units (CFU) per square centimeter of leaf

tissue at six days post infiltration for Xe and Xp;

three days post infiltration for Ps. Error bars

indicate standard deviation. * = p < 0.05, ** = p <

0.01 by Student’s t-test.

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

10.0

FL8000 316.4.1

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

10.0

FL8000 316.4

Wild-type

Log (

CF

U/c

m2)

Wild-type

Wild-type

Roq1

Roq1

Roq1

Log (

CF

U/c

m2)

Log (

CF

U/c

m2)

A

B

C

Xp Xp ∆XopQ

Xe Xe ∆XopQ

Ps Ps ∆HopQ1

* *

* *

*


https://doi.org/10.1101/813758


Figure 2. Bacterial disease symptoms on Roq1 tomato. Xanthomonas perforans 4B (Xp), Xanthomonas

euvesicatoria 85-10 (Xe), and Pseudomonas syringae DC3000 (Ps) wild-type and XopQ/HopQ1 knockout strains

were infiltrated into tomato leaf tissue at low inoculum and disease symptoms were imaged at twelve, thirteen and

four days post infiltration for Xe, Xp and Ps respectively. The infiltration was performed using a needless syringe

and circular wounds from the infiltration are visible. The distal part of region of each leaf was infiltrated and the

proximal part was left untreated. Xe and Xp were infiltrated at an OD600 of 0.0001 whereas Ps was infiltrated at an

OD600 of 0.00005.

Wild-type

Roq1

Xanthomonas perforans 4B∆XopQ

To

ma

to g

en

oty

pe

Wild-typeXanthomonas euvesicatoria 85-10

∆XopQWild-typePseudomonas syringae DC3000

∆HopQ1Wild-type


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Figure 3. Phylogenetic tree of RipB proteins. Two

major clades of RipB alleles from Ralstonia

solanacearum strains are visible in a maximum

likelihood tree generated from XopQ, HopQ1 and RipB

protein sequences. RipB alleles from Ralstonia

solanacearum strains GMI1000 and MolK2 were

cloned for testing in this study and are indicated by

pink dots. Several alleles have truncations that may

make them nonfunctional (indicated by a half circle).

Abbreviations were used for Ralstonia (R) and

Ralstonia solanacearum (Rs).

Rs CFBP3059 (WP_064050487)

Cla

de

1

Rs KACC10722 (WP_075465501)

R. syzygii R24 (CCA88514)

R. BDBR229 (WP_078222314)

Rs PSI07 (WP_013213770)

R. pseudosolanacearum (AST30933)

Rs FJAT-1458 (WP_016722400)

Rs RS1000 (BAD42390)

Rs SD54 (WP_016726261)

Rs (CUV54608)

Rs FQY4 (WP_020830961)

Rs Rs-10-244 (WP_028860117)

Rs YC45 (AKZ27773)

Rs UW757 (WP_064477468)

Rs RD15 (WP_071011882)

Rs (CUV47980)

Rs CMR15 (WP_020749919)

Rs CaRs-MeP (WP_071093460)

Rs Rs-09-161 (WP_028852505)

Rs P824 (WP_058908337)

Rs (CUV28197)

Rs T523 (WP_119889860)

Rs PSS4 (WP_071624165)

Rs CFBP7014 (WP_055325573)

Rs Po82 (WP_014618158)

Rs UW551 (WP_020956993)

Rs UW349 (WP_039572094)

Rs Molk2 (WP_003278485)

Rs CFIA906 (KFZ93252)

Rs UW491 (WP_039559119)

Rs RS489 (WP_097909234)

Rs CFBP2957 (CBJ44424)

Rs B50 (WP_043946231)

Rs CIP120 (WP_064298240)

Rs P597 (WP_064045844)

Rs UW700 (OYQ16135)

Rs BBAC C1 (WP_075456569)

Rs GMI1000 (WP_011000212)

Rs OE1-1 (APC67368)

Rs K60 ID:9545959

Selected for expression

Race 3 Biovar 2 strain

Truncated allele

Rs BK1002 (APC67368)C

lad

e 2


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Figure 4.

Roq1-dependent recognition of RipB in Nicotiana

tabacum. Agrobacterium tumefaciens was used to

transiently expressed XopQ, RipBGMI1000 and RipBMolK2

along with either Roq1 or an empty vector (EV) control

in wild-type Nicotiana tabacum and a roq1 loss of

function mutant. The Agrobacterium was infiltrated at a

total OD600 of 0.5 and the leaves were imaged at three

days post infiltration.

Wild

-type

roq1-1

XopQ

EV

RipBGMI1000

EV

RipBMolK2

EV

Roq1

EV

roq1-1

XopQ

Roq1

RipBGMI1000

Roq1

RipBMolK2

Roq1

Nic

otia

na t

abacum

genoty

pe

Transient expression construct


https://doi.org/10.1101/813758


Figure 5.

Bacterial Wilt disease development in Roq1 tomatoes.

(A) Wild-type and Roq1 tomatoes were infected with

wild-type and RipB mutant Ralstonia solanacearum

strain GMI1000 by soil soak inoculation. Disease

symptoms were monitored over 14 days, with no

wilting corresponding to a Disease Index of 0 and

complete wilting corresponding to a Disease Index of

4. Error bars indicate standard error. (B) Wild-type and

Roq1 tomato plants 8 days after petiole inoculation

with approximately 2,000 cells of wild-type GMI1000.

A

Wild-type + Rs

Wild-type + Rs∆ripB

Roq1 + Rs

Roq1 + Rs∆ripB

B

Wild-type Roq1

B

Days post inoculation

Dis

ease Index


https://doi.org/10.1101/813758


Table 1. Field trial results. A field trial was conducted

in Florida with disease pressure from Xanthomonas

perforans. Disease severity was scored out of 100

using the Horsfall-Barratt scale. Harvested tomatoes

were graded and sized by USDA specifications to

calculate the total marketable yield. The values shown

are means ± standard deviation from at least four

replicate plots of ten plants each. Tomato plants

expressing the Bs2 immune receptor gene were

included as a resistant control.

Season /

Genotype

Disease

severity

Marketable yield

(kg/ha)

Spring 2018

Fla. 8000 86 ± 5 54,655 ± 9,450

Fla. 8000 Roq1 1 ± 1 52,656 ± 3,810

Fla. 8000 Bs2 1 ± 1 66,270 ± 10,309

Fall 2018

Fla. 8000 25 ± 7 19,576 ± 11,038

Fla. 8000 Roq1 5 ± 1 18,538 ± 5,901

Fla. 8000 Bs2 0 ± 0 33,770 ± 13,176

Spring 2019

Fla. 8000 84 ± 2 73,009 ± 15,243

Fla. 8000 Roq1 11 ± 7 92,837 ± 11,072

Fla. 8000 Bs2 5 ± 1 80,516 ± 14,531


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Strain Host(s) Phylotype RipB allele RipB accession

GMI1000 Tomato, Pepper, Arabidopsis I Present WP_011000212

RS1000 Tomato I Present BAD42390

OE1-1 Tobacco I Truncated APC67368

BK1002 Tobacco Truncated LC459955

Y45 Tobacco IB Absent

K60 Tomato, Tobacco IIA Truncated CCF97494

CFBP2957 Tomato IIA Present CBJ44424

Po82 Tomato, Banana, Potato IIB Present WP_014618158

IPO1609* Potato IIB Present WP_020956993

MolK2 Banana IIB Present WP_003278485

UW551* Geranium, Tomato IIB Present EAP72492

CMR15 Tomato III Present WP_020749919

PSI07 Tomato IV Present WP_013213770

BDB R229 Banana Present WP_078222314

Table 2. RipB occurrence and host range in Ralstonia solanacearum. The

published host range of select Ralstonia solanacearum strains is listed along

with the identified RipB allele. Truncated indicates that the identified allele is

missing conserved residues at the C terminus and is putatively non-functional. *

indicates a Race 3 Biovar 2 Select Agent strain.


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Documents

Xanthomonas Pseudomonas syringae and Ralstonia solanacearum · genes (X. Li, Kapos, and Zhang 2015; Jones, Vance, and Dangl 2016; Kapos, Devendrakumar, and Li 2019). While effector